Dissociated neurons from normal and mutant Drosophila larval central nervous system in cell culture

Abstract

A primary dissociated cell culture of Drosophila larval central nervous system is reported. Divisions of neuroblasts and vigorous outgrowth of neurites could be observed in culture. Within 24 hr cultured cells exhibited characteristic neuronal morphology and unimpaired ability to synthesize and accumulate acetylcholine. This cell culture system renders easy access to experimental analysis of normal neuronal properties and the altered mechanisms in neurological mutants. Single-channel currents induced by acetylcholine and regenerative action potentials were studied in the somata of the dissociated neurons. The appearance of Na channels in these cultured neurons was demonstrated by the cell lethality induced by veratridine and inhibition of the effect by tetrodotoxin. Dissociated neurons from a temperature-sensitive paralytic mutant napts, in which nerve conduction fails at high temperature, were studied in culture. Neuronal growth was not affected by this mutation, nor by tetrodotoxin. However, napts neurons showed greatly reduced sensitivity to veratridine even at 21 degrees C, a temperature at which napts individuals behave normally. This finding indicates expression of the napts phenotype at a level of isolated single cells and provides independent evidence that napts affects Na channel function.

The Steroid Hormone 20-Hydroxyecdysone Enhances Neurite Growth ofDrosophilaMushroom Body Neurons Isolated during Metamorphosis

“…The techniques used to prepare and maintain cultures of dissociated Drosophila C NS tissue were adapted from those developed for Drosophila larval neurons (Wu et al, 1983) and Manduca pupal motor neurons (Prugh et al, 1992). Progeny from 201Y ϫ UAS G -lacZ matings were collected as prepupae, sexed, and monitored in a humid 60 mm covered glass Petri dish to determine the time of head eversion, signif ying the start of the pupal phase of metamorphosis (Bainbridge and Bownes, 1981).…”

“…Brain tissue encompassing the M Bs was regionally dissected away from the thoracoabdominal ganglion and optic lobes using minutien pins (Fine Science Tools, Foster C ity, CA). The isolated tissue was incubated for 1 hr at room temperature in freshly prepared enzyme solution of 0.1 mg /ml collagenase (Worthington, Freehold, NJ) and 0.4 mg /ml dispase (Boehringer Mannheim, Indianapolis, I N) in Rinaldini's saline: 137 mM NaC l, 2.68 mM KC l, 0.36 mM NaH 2 PO 4 , 11.9 mM NaHC O 3 , and 5.55 mM glucose (Wu et al, 1983).…”

Voltage clamp analysis of membrane currents in larval muscle fibers of Drosophila: alteration of potassium currents in Shaker mutants

“…More incisive analysis of mutant potassium channels can be achieved by patch recording from cultured Drosophila neurons (Wu et al, 198313;Sun and Wu, 1984).…”

Flow Cytometry and Karyotype Analysis ofD. melanogasterEye Disc Cells

“…Finally, larvae are washed three times in a 100 ml drop of MilliQ water. Single larvae are then transferred in 50 ml drops of Drosophila Modified Medium, (DMM; 27% Leibovitz-15 medium -SIGMA Cat#l518 -, and 73% Saline Solution 5.4 mM CaCl 2 , 21.4 mM KCl, 12.3 mM MgCl 2 , 36 mM NaCl, 4.8 mM NaHCO 3 , 1.5 mM KH 2 PO 4 , 11.1 mM glucose and 24.8 mM Tris pH 7.1) 7 and the eye discs are hand-dissected with a pair of high precision stainless steel tweezers (Dumont n.5). Eye discs are collected at room temperature in a 1.5 ml LowBind tube (Eppendorf ) containing 400 ml of DMM.…”

“…Here, we report a new method adapted from protocols used to produce primary cell cultures, 7 to dissociate eye disc cells and make them suitable for a variety of cytological and molecular analyses. Our method combines a gentle enzymatic treatment of the eye imaginal discs with a mild mechanical separation of the cells.…”